DT-Diaphorose or NAD(P)H:quinone oxidoreductase (NQO1) plays a key role in the deactivation of many environmental quinones and xenobiotics and has been proposed to be important in both chemoprotection and chemoprevention. NQO1 has also attracted considerable attention because of its ability to activate certain bioreductive antitumor quinones and its elevated activity in tumors such as non small cell lung cancer (NSCLC). In an effort to develop new agents for the therapy of NSCLC, we will examine the ability of novel antitumor quinones to serve as substrates for human NQO1 and test the hypothesis that compounds which are efficiently bioactivated by NQO1 are selectively cytotoxic to NSCLC cells with elevated NQO1 activity. It is also critical to understand the mechanisms underlying the elevated expression of NQO1 in NSCLC relative to SCLC. We therefore propose to characterize the specific cis-acting DNA sequences and trans-acting nuclear protein-DNA interactions that mediate NSCLC specific expression of NQO1. We have characterized a polymorphism in NQO1, a homozygous mutation which leads to a total loss of NQO1 protein and enzyme activity, although NQO1 mRNA can readily be detected. We will determine if regulation of the mutant hNQO1 is at the level of translation or protein instability and define the alterations in protein structure which occur as a result of the mutation. Whether NQO1 plays any physiological role or acts on endogenous substrates is as yet unknown. Our data suggests that NQO1 regenerates antioxidant forms of alpha-tocopherol after free radical attack and this observation will be pursued in this application. The potential role of NQO1 as an antioxidant enzyme may also contribute to the chemopreventive role of NQO1. The characterization of a polymorphism in NQO1 which leads to a total loss of NQO1 protein and activity gives us a unique opportunity to examine the association between lack of NQO1 and susceptibility to cancer in a human population. We will examine the prevalence of the NQO1 polymorphism in patients with colon ademomas, an intermediate stage in the development of colon cancer, and matched disease-free individuals. The proposed studies represent an integrated chemical, biochemical and molecular approach to determining the significance of NQO1 for bioreductive drug activation and chemoprevention.